N-terminal ATP-binding Domain Research Articles

Development of Specific Monoclonal Antibodies against Porcine RIG-I-like Receptors Revealed the Species Specificity.

The RIG-I-like receptors (RLRs) play critical roles in sensing and combating viral infections, particularly RNA virus infections. However, there is a dearth of research on livestock RLRs due to a lack of specific antibodies. In this study, we purified porcine RLR proteins and developed monoclonal antibodies (mAbs) against porcine RLR members RIG-I, MDA5 and LGP2, for which one, one and two hybridomas were obtained, respectively. The porcine RIG-I and MDA5 mAbs each targeted the regions beyond the N-terminal CARDs domains, whereas the two LGP2 mAbs were both directed to the N-terminal helicase ATP binding domain in the Western blotting. In addition, all of the porcine RLR mAbs recognized the corresponding cytoplasmic RLR proteins in the immunofluorescence and immunochemistry assays. Importantly, both RIG-I and MDA5 mAbs are porcine specific, without demonstrating any cross-reactions with the human counterparts. As for the two LGP2 mAbs, one is porcine specific, whereas another one reacts with both porcine and human LGP2. Thus, our study not only provides useful tools for porcine RLR antiviral signaling research, but also reveals the porcine species specificity, giving significant insights into porcine innate immunity and immune biology.

AlphaFold predicted structure of the Hsp90-like domains of the neurodegeneration linked protein sacsin reveals key residues for ATPase activity.

The ataxia-linked protein sacsin has three regions of partial homology to Hsp90's N-terminal ATP binding domain. Although a crystal structure for this Hsp90-like domain has been reported the precise molecular interactions required for ATP-binding and hydrolysis are unclear and it is debatable whether ATP biding is compatible with these domains. Furthermore, the Identification of a sacsin domain(s) equivalent to the middle domain of Hsp90 has been elusive. Here we present the superimposition of an AlphaFold structure of sacsin with yeast Hsp90, which provides novel insights into sacsin's structure. We identify residues within the sacsin Hsp90-like domains that are required for ATP binding and hydrolysis, including the putative catalytic arginine residues equivalent to that of the Hsp90 middle domain. Importantly, our analysis allows comparison of the Hsp90 middle domain with corresponding sacsin regions and identifies a shorter lid segment, in the sacsin ATP-binding domains, than the one found in the N-terminal domain of Hsp90. Our results show how a realignment of residues in the lid segment of sacsin that are involved in ATP binding can better match equivalent residues seen in Hsp90, which we then corroborated using molecular dynamic simulations. We speculate, from a structural viewpoint, why some ATP competitive inhibitors of Hsp90 may not bind sacsin, while others would. Together our analysis supports the hypothesis that sacsin's function is ATP-driven and would be consistent with it having a role as a super molecular chaperone. We propose that the SR1 regions of sacsin be renamed as HSP-NRD (Hsp90N-Terminal Repeat Domain; residues 84-324) and the fragment immediately after as HSP-MRD (Hsp90 Middle Repeat Domain; residues 325-518).

The mechanics of DNA mismatch repair sliding clamp progression in the prediction of clinically relevant HsMLH1 missense mutations

Lynch Syndrome (LS), the most common inherited colorectal cancer predisposition, is primarily caused by mutations in the DNA mismatch repair (MMR) genes: HsMSH2, HsMLH1, HsMSH6 and HsPMS2. MMR proteins maintain genomic stability by repairing nucleotide mispairing errors during DNA replication. They are highly conserved members of the MutS (MSH) and MutL (MLH/PMS) proteins that play central roles in directing accurate MMR excision. MSH proteins recognize mismatched nucleotides which triggers ATP binding and formation of a sliding clamp that randomly diffuses along the DNA. Previous biophysical studies in E.coli, showed that EcMutS and EcMutL formed cascading sliding clamps on the mismatched DNA, and defined the intermediary activity of EcMutL, as a signal transmitter between the mismatch recognition and the downstream excision processes. However, the detailed mechanics of the human MLH/PMS proteins has remained a puzzle. Using real-time-single molecule imaging we show that human heterodimeric MMR proteins HsMSH2-HsMSH6 and HsMLH1-HsPMS2 exhibit identical mechanism to their bacterial homologs. HsMSH2-HsMSH6 is required to load the major human MLH/PMS heterodimer onto a DNA containing a single mismatch. In its absence, no visible HsMLH1-HsPMS2 sliding clamps were observed. MLH/PMS proteins contain a disordered region, variable in length, that connects the N-terminal ATP binding domain that interacts with HsMSH2-HsMSH6 with the C-terminal dimerization domain of the protein. Our results indicate that HsMSH2-HsMSH6 provides a platform for HsMLH1-HsPMS2 to wrap around the DNA and form a stable sliding clamp, diffusing rapidly along the DNA. Furthermore, it has been shown that increasing the number of amino acids deleted in the linker domain in EcMutL and S. cerevisae ScMlh1 or ScPms1, initially block the ability to transit roadblocks on the DNA to ultimately, total MMR inhibition. MLH/PMS sliding clamps resemble a flexible donut that encircles the DNA with a relatively large donut hole. We hypothesize that any sequence of amino acids could, in principle, occupy this domain with the condition that it remained malleable and capable of wrapping around the DNA. Therefore, few if any LS pathogenic missense protein variants (MPVs) are expected within the HsMLH1 linker domain. To test this, unique missense protein variants (MPVs) located in the coding sequence of HsMLH1 were analyzed, A clear pattern was found, where non-pathogenic (26) and uncertain (366) variants were distributed ubiquitously. However, the disordered linker region did not contain any pathogenic (71) variants. We conclude that uncertain MPVs located in the linker are unlikely to be pathogenic since it is doubtful that any single amino acid substitution could influence the natural disorder that is essential for linker-domain function. The mechanics of sliding clamp progression solves a significant operational puzzle in MMR and provides explicit predictions for the distribution of pathogenic HsMLH1 missense mutations.

Development of a Cell-Based Ligand-Screening System for Identifying Hsp90 Inhibitors.

Because of its critical roles in regulating cellular signal transduction, the molecular chaperone heat-shock protein 90 (Hsp90) has become a novel therapeutic target for various diseases, including cancer, inflammation, and neurological diseases. However, the lack of methods that allow us to directly evaluate the binding of small molecule ligands to intracellular Hsp90 makes the inhibitor development more difficult. Here, we report a simple cell-based assay system for the Hsp90 inhibitor in live-cell environments. In this strategy, the binding activity of ligands of interest is evaluated by competitive inhibition of ligand-directed N-acyl-N-alkyl sulfonamide (LDNASA) chemistry-mediated Hsp90 labeling. Using several known Hsp90 inhibitors, we demonstrated that our method could easily detect the ligand-binding event of Hsp90 in live cells. Our system is applicable to high-throughput ligand screening, and we discovered a new small molecule candidate that binds to the N-terminal ATP binding domain of Hsp90. These results demonstrate the use of the competitive LDNASA-based approach to directly evaluate ligand activity in live cells and identify potent drug candidates from chemical libraries.

The Hsp90 isoforms from S. cerevisiae differ in structure, function and client range

The molecular chaperone Hsp90 is an important regulator of proteostasis. It has remained unclear why S. cerevisiae possesses two Hsp90 isoforms, the constitutively expressed Hsc82 and the stress-inducible Hsp82. Here, we report distinct differences despite a sequence identity of 97%. Consistent with its function under stress conditions, Hsp82 is more stable and refolds more efficiently than Hsc82. The two isoforms also differ in their ATPases and conformational cycles. Hsc82 is more processive and populates closed states to a greater extent. Variations in the N-terminal ATP-binding domain modulate its dynamics and conformational cycle. Despite these differences, the client interactomes are largely identical, but isoform-specific interactors exist both under physiological and heat shock conditions. Taken together, changes mainly in the N-domain create a stress-specific, more resilient protein with a shifted activity profile. Thus, the precise tuning of the Hsp90 isoforms preserves the basic mechanism but adapts it to specific needs.

Structural and functional mapping of Rtg2p determinants involved in retrograde signaling and aging of Saccharomyces cerevisiae.

In Saccharomyces cerevisiae mitochondrial dysfunction induces retrograde signaling, a pathway of communication from mitochondria to the nucleus that promotes a metabolic remodeling to ensure sufficient biosynthetic precursors for replication. Rtg2p is a positive modulator of this pathway that is also required for cellular longevity. This protein belongs to the ASKHA superfamily, and contains a putative N-terminal ATP-binding domain, but there is no detailed structural and functional map of the residues in this domain that accounts for their contribution to retrograde signaling and aging. Here we use Decomposition of Residue Correlation Networks and site-directed mutagenesis to identify Rtg2p structural determinants of retrograde signaling and longevity. We found that most of the residues involved in retrograde signaling surround the ATP-binding loops, and that Rtg2p N-terminus is divided in three regions whose mutants have different aging phenotypes. We also identified E137, D158 and S163 as possible residues involved in stabilization of ATP at the active site. The mutants shown here may be used to map other Rtg2p activities that crosstalk to other pathways of the cell related to genomic stability and aging.

Thermodynamic Analysis of the Geldanamycin-Hsp90 Interaction in a Whole Cell Lysate Using a Mass Spectrometry-Based Proteomics Approach.

Geldanamycin is a natural product with well-established and potent anti-cancer activities. Heat shock protein 90 (Hsp90) is the known target of geldanamycin, which directly binds to Hsp90's N-terminal ATP binding domain and inhibits Hsp90's ATPase activity. The affinity of geldanamycin for Hsp90 has been measured in multiple studies. However, there have been large discrepancies between the reported dissociation constants (i.e., Kd values), which have ranged from low nanomolar to micromolar. Here the stability of proteins from rates of oxidation (SPROX) technique was used in combination with an isobaric mass tagging strategy to measure the binding affinity of geldanamycin to unpurified Hsp90 in an MCF-7 cell lysate. The Kd values determined here were dependent on how long geldanamycin was equilibrated with the lysate prior to SPROX analysis. The Kd values determined using equilibration times of 0.5 and 24 h were 1 and 0.03 μM, respectively. These Kd values, which are similar to those previously reported in a geldanamycin-Hsp90 binding study that involved the use of a fluorescently labeled geldanamycin analogue, establish that the slow-tight binding behavior previously observed for the fluorescently labeled geldanamycin analogue is not an artifact of the fluorescent label, but rather an inherent property of the geldanamycin-Hsp90 binding interaction. The slow-tight binding property of this complex may be related to time-dependent conformational changes in Hsp90 and/or to time-dependent chemical changes in geldanamycin, both of which have been previously proposed to explain the slow-tight binding behavior of the geldanamycin-Hsp90 complex. Graphical Abstract ᅟ.

NMR structural characterization of the N-terminal active domain of the gyrase B subunit from Pseudomonas aeruginosa and its complex with an inhibitor

The N-terminal ATP binding domain of the DNA gyrase B subunit is a validated drug target for antibacterial drug discovery. Structural information for this domain (pGyrB) from Pseudomonas aeruginosa is still missing. In this study, the interaction between pGyrB and a bis-pyridylurea inhibitor was characterized using several biophysical methods. We further carried out structural analysis of pGyrB using NMR spectroscopy. The secondary structures of free and inhibitor bound pGyrB were obtained based on backbone chemical shift assignment. Chemical shift perturbation and NOE experiments demonstrated that the inhibitor binds to the ATP binding pocket. The results of this study will be helpful for drug development targeting P. aeruginosa.

Eukaryotic initiation factor 4AI interacts with NS4A of Dengue virus and plays an antiviral role

Dengue virus (DENV) is a mosquito-borne flavivirus that causes the most prevalent diseases in tropical and subtropical regions. DENV utilizes host factors to facilitate its replication, while host cells intend to restrain virus replication. NS4A of DENV has been implicated to play a crucial role during viral replication. To identify more cellular proteins that are recruited by NS4A, we carried out a tandem affinity purification assay. The mass spectrometry data revealed that human eukaryotic initiation factor 4AI (eIF4AI) was one of potential NS4A-interacting partners. Co-immunoprecipitation data confirmed the interaction between NS4A and eIF4AI, and both the N-terminal ATP-binding domain and C-terminal helicase domain of eIF4AI were involved in their association. Functionally, silencing of eIF4AI by RNAi significantly increased the replication level of DENV1, DENV2 and DENV3. And knockdown of eIF4AI markedly attenuated the phosphorylation of protein kinase regulated by dsRNA-activated (PKR) and eIF2ɑ induced by DENV2 infection. Collectively, these data suggested that a potential role of NS4A in antagonizing host antiviral defense is by recruiting eIF4AI and escaping the translation inhibition mediated by PKR.

Gambogic acid inhibits Hsp90 and deregulates TNF-α/NF-κB in HeLa cells

Gambogic acid (GB) is an important anti-cancer drug candidate, but the target protein by which it exerts its anti-cancer effects has not been identified. This study is the first to show that GB inhibits heat shock protein 90 (Hsp90) and down-regulates TNF-α/NF-κB in HeLa cells. The effects of GB on Hsp90 were studied by characterizing its physical interactions with Hsp90 upon binding, the noncompetitive inhibition of Hsp90 ATPase activity, and the degradation of Hsp90 client proteins (i.e., Akt, IKK) in HeLa cells. GB seems to bind to the N-terminal ATP-binding domain of Hsp90. Additionally, GB suppresses the activation of TNF-α/NF-κB and decreases XIAP expression levels and the ratio of Bcl-2/Bax, which in turn induces HeLa cell apoptosis. Thus, GB represents a promising therapeutic agent for cancer; it may also be useful as a probe to increase understanding of the biological functions of Hsp90.

Structure of the ATP‐binding domain of Plasmodium falciparum Hsp90

Hsp90 is an important cellular chaperone and attractive target for therapeutics against both cancer and infectious organisms. The Hsp90 protein from the parasite Plasmodium falciparum, the causative agent of malaria, is critical for this organism's survival; the anti-Hsp90 drug geldanamycin is toxic to P. falciparum growth. We have solved the structure of the N-terminal ATP-binding domain of P. falciparum Hsp90, which contains a principal drug-binding pocket, in both apo and ADP-bound states at 2.3 A resolution. The structure shows that P. falciparum Hsp90 is highly similar to human Hsp90, and likely binds agents such as geldanamycin in an identical manner. Our results should aid in the structural understanding of Hsp90-drug interactions in P. falciparum, and provide a scaffold for future drug-discovery efforts.

ATPase Domain and Interdomain Linker Play a Key Role in Aggregation of Mitochondrial Hsp70 Chaperone Ssc1

The co-chaperone Hep1 is required to prevent the aggregation of mitochondrial Hsp70 proteins. We have analyzed the interaction of Hep1 with mitochondrial Hsp70 (Ssc1) and the determinants in Ssc1 that make it prone to aggregation. The ATPase and peptide binding domain (PBD) of Hsp70 proteins are connected by a linker segment that mediates interdomain communication between the domains. We show here that the minimal Hep1 binding entity of Ssc1 consists of the ATPase domain and the interdomain linker. In the absence of Hep1, the ATPase domain with the interdomain linker had the tendency to aggregate, in contrast to the ATPase domain with the mutated linker segment or without linker, and in contrast to the PBD. The closest homolog of Ssc1, bacterial DnaK, and a Ssc1 chimera, in which a segment of the ATPase domain of Ssc1 was replaced by the corresponding segment from DnaK, did not aggregate in Delta hep1 mitochondria. The propensity to aggregate appears to be a specific property of the mitochondrial Hsp70 proteins. The ATPase domain in combination with the interdomain linker is crucial for aggregation of Ssc1. In conclusion, our results suggest that interdomain communication makes Ssc1 prone to aggregation. Hep1 counteracts aggregation by binding to this aggregation-prone conformer.

Assays for Identification of Hsp90 Inhibitors and Biochemical Methods for Discriminating their Mechanism of Action

The Hsp90-dependence of many oncogenic proteins has precipitated a great deal of interest in Hsp90 as a drug target, as evidence mounts that Hsp90 inhibitors may be effective chemotherapeutic agents for the treatment of cancer. In addition, Hsp90-inhibitors have shown promise for the treatment of neurodegenerative diseases characterized by the accumulation of toxic denatured protein aggregates. The development of assays for the identification of novel Hsp90 inhibitors began in earnest when it became apparent that the Hsp90 inhibitors available at the time had less than ideal pharmacological properties. This review summarizes what is known about Hsp90's structure and function, its ATPase cycle, its interactions with co-chaperones and clients, and the effect of Hsp90-inhibitors on these processes. It further summarizes various high throughput assays (and secondary confirmatory assays) developed to identify new Hsp90 inhibitors from chemical libraries based on the inhibitors ability to: inhibit Hsp90's ATPase activity; compete for ligand binding to Hsp90 and its N-terminal ATP-binding domain; inhibit Hsp90-dependent refolding of denatured luciferase; and deplete culture cells of Hsp90-dependnet client protein or induce Hsp70 expression. In addition, in vitro assays are described that help determine the site of inhibitor binding to Hsp90 (N- or C-terminal domain) and there mechanism of action based on effects of inhibitors on Hsp90/ co-chaperone and client interactions, and Hsp90 conformation characterized by proteolytic fingerprinting.

The Charged Linker Region Is an Important Regulator of Hsp90 Function

Hsp90 is an ATP-dependent molecular chaperone which assists the maturation of a large set of target proteins. Members of the highly conserved Hsp90 family are found from bacteria to higher eukaryotes, with homologues in different organelles. The core architecture of Hsp90 is defined by the N-terminal ATP binding domain followed by the middle domain and the C-terminal dimerization domain. A long, highly charged linker between the N-terminal domain and the middle domain is a feature characteristic for Hsp90s of eukaryotic organisms. We set out to clarify the function of this linker by studying the effects of deletions in this region in vivo and in vitro. Here we show that increasing deletions in the charged linker region lead to defects ranging from mild temperature sensitivity to a lethal phenotype. The lethal deletion variants investigated in this study still exhibit ATPase activity. Deletion of the charged linker ultimately causes a loss of Hsp90 regulation by co-chaperones, as the sensitivity for Aha1-mediated ATPase acceleration declines, and binding of p23/Sba1 is lost in non-viable deletion constructs. In vivo client assays additionally demonstrated that the deletion of the linker had a pronounced effect on the ability of Hsp90 to facilitate client activation. A partial reconstruction of the linker sequence showed that the supplementation by artificial sequences can rescue the functionality of Hsp90 and restore the conformational flexibility of the protein, required for the processing of client proteins.

Structural–Thermodynamic Relationships of Interactions in the N-Terminal ATP-Binding Domain of Hsp90

Despite its importance as a target in anti-cancer therapeutics and the numerous rational-based inhibitor design efforts aimed at it, there are only limited data available on structural-thermodynamic relationships of interactions of the N-terminal ATP-binding domain of Hsp90 (N-Hsp90). Here, we redress this by presenting an investigation of binding of nucleotides and ansamycin compounds to this domain. Interactions of nucleotides with N-Hsp90 are relatively weak (>10 μM) and are strongly enthalpy driven over the temperature range 10–25 °C. Geldanamycin (GA) and its analogues 17-AAG [17-(allylamino)-17-demethoxy-GA] and 17-DMAG (17-N,N-dimethylaminoethylamino-17-demethoxy-GA) bind more strongly and have a dominant favourable enthalpic contribution over the temperature range investigated. We investigated the temperature dependence of the enthalpic contribution to binding. We found that while the ansamycin compounds have the commonly observed negative value, the nucleotides show a negligible or even a positive ΔCp of binding. These data represent the first observation of a single binding site for which interactions with different ligands result in both negative and positive ΔCp values. By addressing the likely impact of the potential contributions from protein–ligand interactions, we are able to attribute the anomalous ΔCp for the nucleotides largely to a change in the conformation of the domain structure and local motion in the lid region of N-Hsp90 with the concomitant exposure of hydrophobic amino acid side chains.

Single Molecule Investigations of HSP70 Proteins

Heat Shock Proteins 70 (HSP70) are a class of proteins involved in protein folding and are highly upregulated when a cell is under stress. The protein consists of three functional domains: the N-terminal ATP binding domain where ATP and ADP can bind, the substrate binding domain which can bind small residues and the C-terminal domains which acts as a lid for the substrate binding domain. It is thought that the binding of ATP/ADP drives conformational changes in this protein.Using burst analysis with pulsed interleaved excitation, we have performed single-pair Forster resonance energy transfer (FRET) experiments to investigated the distribution of conformations in HSP70 molecules under different conditions. The FRET efficiency is very sensitive to the distance between donor and acceptor on the scale of 2-10 nm and thus provides information over the conformation of the different HSP70 domains as they diffusion through the focus of our confocal microscope. As experiments are performed on single molecules, subpopulations can be directly observed. We have investigated the conformation of HSP70 by cloning a number of mutants that allowed specific labeling of the different domains. Experiments were performed with or without ATP, ADP, and nonhydrolysable ATP analogs as well as in the absence or presence of peptide substrates or cochaperons. We will present how the conformation and flexibility of HSP70 is influenced by these various interactions.

Conserved Conformational Changes in the ATPase Cycle of Human Hsp90

The dimeric molecular chaperone Hsp90 is required for the activation and stabilization of hundreds of substrate proteins, many of which participate in signal transduction pathways. The activation process depends on the hydrolysis of ATP by Hsp90. Hsp90 consists of a C-terminal dimerization domain, a middle domain, which may interact with substrate protein, and an N-terminal ATP-binding domain. A complex cycle of conformational changes has been proposed for the ATPase cycle of yeast Hsp90, where a critical step during the reaction requires the transient N-terminal dimerization of the two protomers. The ATPase cycle of human Hsp90 is less well understood, and significant differences have been proposed regarding key mechanistic aspects. ATP hydrolysis by human Hsp90alpha and Hsp90beta is 10-fold slower than that of yeast Hsp90. Despite these differences, our experiments suggest that the underlying enzymatic mechanisms are highly similar. In both cases, a concerted conformational rearrangement involving the N-terminal domains of both subunits is controlling the rate of ATP turnover, and N-terminal cross-talk determines the rate-limiting steps. Furthermore, similar to yeast Hsp90, the slow ATP hydrolysis by human Hsp90s can be stimulated up to over 100-fold by the addition of the co-chaperone Aha1 from either human or yeast origin. Together, our results show that the basic principles of the Hsp90 ATPase reaction are conserved between yeast and humans, including the dimerization of the N-terminal domains and its regulation by the repositioning of the ATP lid from its original position to a catalytically competent one.

Hsp70 genes in the human genome: Conservation and differentiation patterns predict a wide array of overlapping and specialized functions

BackgroundHsp70 chaperones are required for key cellular processes and response to environmental changes and survival but they have not been fully characterized yet. The human hsp70-gene family has an unknown number of members (eleven counted over ten years ago); some have been described but the information is incomplete and inconsistent. A coherent body of knowledge encompassing all family components that would facilitate their study individually and as a group is lacking. Nowadays, the study of chaperone genes benefits from the availability of genome sequences and a new protocol, chaperonomics, which we applied to elucidate the human hsp70 family.ResultsWe identified 47 hsp70 sequences, 17 genes and 30 pseudogenes. The genes distributed into seven evolutionarily distinct groups with distinguishable subgroups according to phylogenetic and other data, such as exon-intron and protein features. The N-terminal ATP-binding domain (ABD) was conserved at least partially in the majority of the proteins but the C-terminal substrate-binding domain (SBD) was not. Nine proteins were typical Hsp70s (65–80 kDa) with ABD and SBD, two were lighter lacking partly or totally the SBD, and six were heavier (>80 kDa) with divergent C-terminal domains. We also analyzed exon-intron features, transcriptional variants and protein structure and isoforms, and modality and patterns of expression in various tissues and developmental stages. Evolutionary analyses, including human hsp70 genes and pseudogenes, and other eukaryotic hsp70 genes, showed that six human genes encoding cytosolic Hsp70s and 27 pseudogenes originated from retro-transposition of HSPA8, a gene highly expressed in most tissues and developmental stages.ConclusionThe human hsp70-gene family is characterized by a remarkable evolutionary diversity that mainly resulted from multiple duplications and retrotranspositions of a highly expressed gene, HSPA8. Human Hsp70 proteins are clustered into seven evolutionary Groups, with divergent C-terminal domains likely defining their distinctive functions. These functions may also be further defined by the observed differences in the N-terminal domain.

Effectiveness of Hsp90 Inhibitors as Anti-Cancer Drugs

Hsp90 is a chaperone with over 100 identified client proteins. What makes Hsp90 especially promising as a target for anti-cancer drugs is that many of its client proteins are in signaling and chromatin-remodeling pathways, and these pathways are often disrupted in many types of cancers. Recently, it was determined that Hsp90 bound to a client protein in a co-chaperone complex has a higher ATPase activity and binds to the geldanamycin inhibitor with over 100-fold higher affinity than the low-ATPase form. Consequently, despite Hsp90 being an abundant protein in most cell types, Hsp90 inhibitors accumulate at high levels primarily in tumor cells because tumor cells are "oncogene addicted" and require especially high levels of the high-ATPase form of Hsp90. Numerous classes of Hsp90 inhibitors have recently been developed, such as the anasamysin geldanamycin and derivatives 17-AAG and 17-DMAG; the macrolide radicicol and derivatives; purine-scaffold derivatives; pyrazoles; and shepherdins that bind to the N-terminal high-affinity ATP-binding domain of Hsp90. Other inhibitors have recently been shown to bind to the C-terminal dimerization domain of Hsp90, such as cisplatin and novobiocin, or modify Hsp90 postranslationally, such as histone deacetylase or proteasome inhibitors. In this mini-review, we present hypothetical mechanisms for Hsp90 inhibitors in treating cancers, preliminary studies in early clinical trials, and potential tumor-killing and tumor-promoting activities of Hsp90 inhibitors.

Crystal structures of human HSP90α-complexed with dihydroxyphenylpyrazoles

A series of dihydroxyphenylpyrazole compounds were identified as a unique class of reversible Hsp90 inhibitors. The crystal structures for two of the identified compounds complexed with the N-terminal ATP binding domain of human Hsp90α were determined. The dihydroxyphenyl ring of the compounds fits deeply into the adenine binding pocket with the C2 hydroxyl group forming a direct hydrogen bond with the side chain of Asp93. The pyrazole ring forms hydrogen bonds to the backbone carbonyl of Gly97, the hydroxyl group of Thr184 and to a water molecule, which is present in all of the published HSP90 structures. One of the identified compounds (G3130) demonstrated cellular activities (in Her-2 degradation and activation of Hsp70 promoter) consistent with the inhibition of cellular Hsp90 functions.